The present invention relates to the use of lipid dispersions to deliver biologically active substances to cells. In particular, the present invention relates to emulsion and micellar formulations and to the ability of these formulations to form stable complexes with biologically active substances and thereby facilitate the delivery of these substances to cells.
Cationic liposomes are of interest as a non-viral vehicle for the delivery of biologically active substances such as drugs, hormones, enzymes, nucleic acids and antigens, including viruses, to cells both in vitro and in vivo. Indeed, cationic liposomes have been demonstrated to deliver genes in vivo (Nabel, E. G., et al. (1990) Science, 249: 1285-1288), (Brigham, K. L., et al. (1989) Am. J. Respir. Cell Mol. Biol., 195-200, Stribling, R., et al. (1992) Proc. Natl. Acad. Sci. U.S.A., 89: 11277-11281), (Plautz, G. E., et al. (1993) Proc. Natl. Acad. Sci. U.S.A., 90: 4645-4649, Stewart, M. J., et al. (1992) Hum. Gene Ther., 3: 267-275). However, the inhibition by serum components of the transfer of nucleic acids by cationic liposomes limits the application of liposomes as a vector for nucleic acids in vivo to regional administrations which avoid exposure to serum.
In addition, stability is a major problem limiting the use of liposomes, both in terms of shelf life and after administration in vivo. Thus, it is desirable to explore the use of other types of lipid dispersions as delivery systems utility for biologically active substances.
U.S. Pat. No. 4,610,888 refers to the use as a drug-delivery system of water-in-oil emulsions in which the volume of aqueous phase ranges from about 0.7% to about 10.25% of the volume of the lipid components used. However, such water-in-oil emulsions are unsuitable for delivering substances in blood or in other aqueous body tissues.
The present invention relates to novel emulsion and micellar formulations useful for delivering biologically active substances to cells. The emulsion and micellar formulations of this invention are compatible with blood, retain activity in the presence of serum and are stable in storage. The emulsions of this invention comprise lipid components and an aqueous carrier, where the lipid components comprise an oil component, a cationic amphiphile component, preferably a cationic lipid, and a nonionic surfactant component. The micellar formulations comprise lipid components and an aqueous carrier, where the lipid components comprise a cationic amphiphile component and a nonionic surfactant component. The lipid components of the emulsion and micellar formulation of the present invention may further comprise a neutral phospholipid component.
xe2x80x9cComponentxe2x80x9d as used throughout the specification and claims is defined as: comprising at least one cationic amphiphile or a mixture of amphiphiles when used in the phrase xe2x80x9camphiphile componentxe2x80x9d; comprising at least one oil or a mixture of oils when used in the phrase xe2x80x9coil componentxe2x80x9d; comprising at least one nonionic surfactant or a mixture of nonionic surfactants when used in the phrase xe2x80x9cnonionic surfactant componentxe2x80x9d; comprising at least one neutral phospholipid or a mixture of neutral phospholipids when used in the phrase xe2x80x9cneutral phospholipid componentxe2x80x9d.
The invention further relates to complexes formed by combining biologically active substances and the above-identified emulsion and micellar formulations. These biologically active substance:emulsion and biologically active substance:micelle complexes are stable over time and may have therapeutic and/or prophylactic utility in vivo depending on the activity of the biologically active substance contained in the complex.
This invention also provides a method for delivering a biologically active substance to cells by exposing cells to the complexes of this invention. In one embodiment, a method of exposing cells to a biologically active substance is provided, said method comprising culturing said cells in the presence of a biologically active substance:emulsion complex or a biologically active substance:micelle complex thereby facilitating delivery of the biologically active substance to cells.
The invention further provides a method of delivering a biologically active substance to cells in vivo comprising administering to an animal or human the complexes of this invention. It is to be understood that the complexes used for the delivery of biologically active substances to cells in vitro or in vivo may be freshly prepared by admixture or may be prepared earlier and stored prior to their use.
The invention further relates to a kit containing an emulsion or micellar formulation of the present invention. The invention also provides a kit containing a complex formed between a biologically active substance and an emulsion or micellar formulation of the present invention.
Methods for producing emulsion and micellar formulations according to the invention are also provided herein.
In one embodiment, a method for producing an emulsion formulation of this invention comprises
a) combining an organic solvent with an oil component, a cationic amphiphile component and a nonionic surfactant component;
b) removing the organic solvent to leave a lipid film; and
c) suspending the lipid film in an aqueous carrier to produce said emulsion formulation miscible in aqueous solution.
In an alternative embodiment, the oil may serve as the organic solvent in step (a) such that the method for producing an emulsion formulation of this invention comprises
a) combining an oil component, a cationic amphiphile component and a nonionic surfactant component; and
b) adding an aqueous carrier to the combination of step (a).
When a neutral phospholipid component is to be included in the emulsion, the neutral phospholipid component is combined with the above components in step (a).
The method for producing a micellar formulation miscible in aqueous solution comprises:
a) combining an organic solvent with a cationic amphiphile component and a nonionic surfactant component;
b) removing the organic solvent to leave a lipid film; and
c) suspending the lipid film in an aqueous carrier to produce said micellar formulation miscible in aqueous solution.
When a neutral phospholipid component is to be included in the micellar formulation, the neutral phospholipid component is combined with the above components in step (a).